Plant growth is accomplished by orderly cell division and tightly regulated cell expansion. In plants, the contribution of cell expansion to growth is of much greater significance than in most other organisms; all plant organs owe their final size to a period of significant cell elongation, which usually follows active cell division. Further, the sessile nature of plants requires that they make fine but responsive adjustments in growth to survive harsh environmental conditions and to optimize their use of limited resources (Trewavas (1986) “Resource allocation under poor growth conditions: A major role for growth substances in developmental plasticity” In Plasticity in Plants, D. H. Jennings and A. J. Trewavas, eds (Cambridge, UK: Company of Biologists Ltd.), pp. 31–76).
In Arabidopsis, cell elongation is largely responsible for hypocotyl growth in germinating seedlings and extension of inflorescences (bolting) at the end of vegetative growth. Coordinate control of plant growth is regulated by both external stimuli and internal mechanisms. Of the external signals, the most obvious is light (Deng, X.-W. (1994) Cell 76:423–426). Light inhibits hypocotyl elongation and promotes cotyledon expansion and leaf development in seedlings, and photoperiod is crucial for flower initiation in a large number of species.
The internal components of plant signaling are generally mediated by chemical growth regulators (phytohormones; reviewed in Klee, H., and Estelle, M. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:529–551). Thus, plant growth in response to environmental factors is modulated by plant hormones acting alone or in concert (Evans “Functions of hormones at the cellular level of organization” In Hormonal Regulation of Plant Physiology, T. K. Scott, ed (Berlin: Springer-Verlag), pp. 23–79), and growth depends on regulated cellular events, such as division, elongation, and differentiation.
Gibberellic acid (GA) and cytokinins promote flowering; in addition, GA stimulates stem elongation, whereas cytokinins have the opposite effect, reducing apical dominance by stimulating increased axillary shoot formation. Conversely, auxins promote apical dominance and stimulate elongation by a process postulated to require acidification of the cell wall by a K−-dependent H+-pumping ATPase (Rayle, D. L., and Cleland, R. E. (1977) Curr. Top. Dev. Biol. 11:187–214).
In addition to the classic hormones, such as auxin and gibberellic acid (GA), brassinosteroids (BRs) have been discovered to be important in growth promotion (reviewed in Clouse (1996) Plant J. 10:1–8). The most recently discovered class of plant growth substances, the BRs, has been to date the least studied; however, rapid progress toward understanding BR biosynthesis and regulation is now being made (Yokota, T. (1997) Trends Plant Sci. 2:137–143). The term BRs collectively refers to the growth-promoting steroids found in plants (Grove et al. (1979) Nature 281:216–217). They are structurally very similar to the molting hormones of insects, ecdysteroids (Richter and Koolman (1991) “Antiecdysteroid effects of brassinosteroids in insects” in Brassinosteroids: Chemistry, Bioactivity, and Applications, H. G. Cutler, T. Yokota, and G. Adam, eds (Washington, D.C.: American Chemical Society), pp. 265–279), but active BRs have unique structural features. As shown in FIG. 1, a 6-oxolactone or 7-oxalactone in the B ring, 5αhydrogen, and multiple hydroxylations at four different positions with specific stereochemistry have been proposed as an essential configuration for BRs (reviewed in Marquardt and Adam (1991) “Recent advances in brassinosteroid research” in Chemistry of Plant Protection, W. Ebing, ed (Berlin: Springer-Verlag), pp. 103–139). Among >40 naturally occurring BRs, brassinolide (BL; 2α, 3α, 22(R), 23(R)-tetrahydroxy-24(S)-methyl-B-homo-7-oxa-5α-cholestan-6-one) has been shown to be the most biologically active (reviewed in Mandava (1988) Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:23–52). As a major biological effect, BRs stimulate longitudinal growth of young tissues via cell elongation and cell division (reviewed in Clouse (1996), supra; Fujioka and Sakurai (1997a) Nat. Prod. Rep. 14:1–10).
Elucidating the BR biosynthetic pathways has been a major area of recent interest. Biochemical analyses have been used to elucidate the BR biosynthetic pathway (Fujioka et al. (1996) Plant Cell Physiol. 37:1201–1203; Choi et al. (1997), Phytochemistry 44:609–613), and mutational analyses are being used to confirm this pathway. Similar to the biosynthetic pathways of the human steroid hormones and insect ecdysteroids (Rees (1985) “Biosynthesis of ecdysone” in Comprehensive Insect Physiology, Biochemistry and Pharmacology, G. A. Kerkut and L. I. Gilbert, eds (Oxford, UK: Pergamon Press), pp. 249–293; Granner, D. K. (1996) “Hormones of the gonads” in Harper's Biochemistry, R. K. Murray, D. K. Granner, P. A. Mayes, and V. W. Rodwell, eds (Stamford, Conn.: Appleton and Lange Press), pp. 566–580), BRs are synthesized via multiple parallel pathways (Fujioka et al. (1996) Plant Cell Physiol. 37:1201–1203; Choi et al. (1997), supra). Starting from the initial precursor, campesterol (CR), the BR intermediates undergo a series of hydroxylations, reductions, an epimerization, and a Baeyer-Villigerutype oxidation leading to the most oxidized form, BL (Fujioka and Sakurai (1997b) Physiol. Plant. 100:710–715; FIG. 1). Castasterone (CS) oxidation, the last step in BR biosynthesis, is not found in some species, such as mung bean. In that case, CS plays a role as the major BR rather than BL (Yokota et al. (1991) “Metabolism and biosynthesis of brassinosteroids” in Brassinosteroids: Chemistry, Bioactivity, and Application, H. G. Cutler, T. Yokota, and G. Adam, eds (Washington, D.C.: American Chemical Society), pp. 86–96). Traditionally, BR biosynthetic pathways have been elucidated by feeding deuterio-labeled intermediates to BR-producing cell lines of Madagascar periwinkle (Sakurai and Fujioka (1996) “Catharanthus roseus (Vinca rosea): In vitro production of brassinosteroids” in Biotechnology in Agriculture and Forestry, Y. P. S. Bajaj, ed (Berlin: Springer-Verlag), pp. 87–96.). The present model, including parallel branched pathways and early and late C-6 oxidation pathways, was established using these feeding studies (Fujioka and Sakurai (1997a), supra, Fujioka and Sakurai (1997b), supra; Sakurai and Fujioka (1997) Biosci. Biotechnol. Biochem. 61:757–762).
Although the brassinosteriod system is a less well understood class of plant growth substances (BRs; Mitchell, et al. (1970) Nature 225:1065–1066; Grove et al. (1979) Nature 281:216–217; Mandava, N. B. (1988) Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:23–52), several such compounds have been identified and are known to effect elongation of cells in various plant tissues, their biosynthesis, regulation, and mechanism of action have only recently begun to be elucidated (reviewed in Clouse, S. D. (1996) Plant J. 10:1–8; Fujioka, S., and Sakurai, A. (1997) Physiol. Plant. 100:710–715).
Several types of dwarf or dwarflike mutants have been described in Arabidopsis. A number of mutations have been identified that affect either light-dependent (cop, det, and fusca [fus; another group of mutants with some members perturbed in light-regulated growth]) or hormone signaling (axr2) pathways and whose pleiotropic phenotypes include defects in cell elongation. The majority of these mutants also have other alterations in their phenotypes. At least five GA mutants have been described as being reduced in stature (Koornneef and Van der Veen (1980) Theor. Appl. Genet. 58:257–263). GA biosynthetic mutants may also have no or defective flower development and are marked by an absence of viable pollen. Reduced levels of endogenous gibberellins are also a characteristic (Barendse et al.(1986) Physiol. Plant. 67:315–319; Talon et al. (1990) Proc. Natl. Acad. Sci. USA 87:7983–7987), and their phenotype can be nearly restored to that of the wild type by the addition of exogenous GA. (Koornneef and Van der Veen (1980) Theor. Appl. Genet. 58:257–263).
Another hormone mutation, auxin resistant2 (axr2), results in plants with a dwarf phenotype both in the light and in darkness as well as increased resistance to high levels of auxin, ethylene, and abscisic acid (Timpte et al. (1992) Planta 188:271–278). An interesting relationship exists between light regulation and cytokinin levels. Arabidopsis seedlings grown in the dark in the presence of cytokinins have open cotyledons, initiate chloroplast differentiation and leaf development, and activate transcription from the chlorophyll a/b binding protein gene (CAB) promoter. Importantly, they also display a cytokinin dose-dependent dwarf phenotype.
Dwarf Arabidopsis mutants that are rescued by addition of BRs have also been described (Kauschmann et al. (1996) Plant J. 9:701–713; Li et al. (1996) Science 272:398–401; Szekeres et al. (1996) Cell 85:171–182; Azpiroz et al. (1998) Plant Cell 10:219–230), including the following three mutants: dwarf1 (dwf1; Kauschmann et al. (1996) Plant J. 9:701–713), constitutive photomorphogenesis and dwarfism (cpd; Szekeres et al. (1996) Cell 85:171–182), and det2 (Li et al. (1996) Science 272:398–401). These mutants have been shown to be defective in steroid biosynthesis. DWF1 (Feldmann et al. (1989) Science 243:1351–1354) was cloned first (GenBank accession number U12400). Takahashi et al. (1995) Genes Dev. 9:97–107 hypothesized that DWF1, which they isolated with an allele of dwf1, referred to as diminuto1 (dim1), contains a potential nuclear targeting signal, which may confer a regulatory function to the protein. However, Mushegian and Koonin (1995) Protein Sci. 4:1243–1244 indicated that DWF1 displays limited homology with flavin adenine dinucleotide (FAD) independent oxidoreductase, suggesting an enzymatic function in BR biosynthesis. According to Kauschmann et al. (1996), supra (dwf1–6 described as cabbage1 [cbb1]), dwf1 mutants were rescued by exogenous application of BRs.
DET2 was shown to encode a putative steroid 5α-reductase, mediating an early step in BR biosynthesis (Li et al. (1996), supra, Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554–3559; Fujioka et al. (1997) Plant Cell 9:1951–1962; FIG. 1). Moreover, det1 and det2 have a decreased requirement for cytokinins in tissue culture and appear to be saturated for a cytokinin-dependent delay in senescence (Chory et al. (1994) Plant Physiol. 104:339–347). CPD has been proposed to be a novel cytochrome P450 (CYP90A1; Szekeres et al. (1996), supra), encoding a putative 23α-hydroxylase that acts in BR biosynthesis. The range of phenotypes in the deetiolated (det) and constitutive photomorphogenic (cop) light-regulatory mutants is broad. Mutations in DET1, COP1, COP8, COP9, COP10, and COP11 result in constitutive derepression of substantial portions of the photomorphogenic program (Chory, et al. (1989b) Cell 58:991–999; Deng, X.-W., and Quail, P. H. (1992) Plant J. 2:83–95; Wei, N., and Deng, X.-W. (1992) Plant Cell 4:1507–1518; Wei et al. (1994) Plant Cell 6:629–643), whereas mutations in COP4 seem to affect only morphology and gene expression (Hou et al. (1993) Plant Cell 5:329–339). The only invariant phenotype in this class of light-regulatory mutants is a substantial reduction in height in both light and darkness.
There are additional dwarfs that are insensitive to one of these hormones, such as bri (brassinosteroid insensitive; Clouse et al. (1996) Plant Physiol. 111:671–678; Li and Chory (1997) Cell 90:929–938), gai (gibberellic acid insensitive; Koornneef et al. (1985) Physiol. Plant. 65:33–39), and axr2 (auxin resistant2; Timpte et al. (1994) Genetics 138:1239–1249). Clouse et al. (1996), supra isolated bri by screening ethyl methanesulfonate-mutagenized populations for mutants whose root growth is not retarded at inhibitory concentrations of BR. Thus, the BRI protein is proposed to be involved in BR signal perception or transduction (Clouse (1996), supra). Kauschmann et al. (1996), supra described a phenotypically similar mutant cbb2 that maps to the same location. In addition, the dwf2 alleles possess a phenotype similar to bri and map to the same region (Feldmann and Azpiroz (1994) “dwarf (dwf) and twisted dwarf (twd)” in Arabidopsis: An Atlas of Morphology and Development, J. Bowman, ed (New York: Springer-Verlag), pp. 82–85). It seems likely that all of the BR-insensitive dwarf mutants described to date are allelic. Recently, BRI has been cloned and shown to encode a leucine-rich-repeat receptor kinase, suggesting a role in the BR signal transduction pathway (Li and Chory (1997), supra).
Mutants defective in BR biosynthesis have also been isolated in other plant species. Bishop et al. (1996) Plant Cell 8:959–969 isolated a tomato dwarf mutant by transposon tagging. The tomato Dwarf gene encodes a pioneering member of the CYP85 family, and it appears to be involved in BR biosynthesis. In addition, Nomura et al. (1997) Plant Physiol. 113:31–37 reported that the lka and lkb mutants in garden pea are deficient in BR biosynthesis (lkb) or perception (lka).
Currently, little is known about the downstream events that occur in response to these signals and thereby directly control cell size. This is because the biochemical and cell biological processes involved have thus far been difficult to address. In addition, there is little information about the integration of regulatory signals converging at the cell from different signaling pathways and the ways they are coordinately controlled. In particular, the interaction of light and hormones in the control of cell elongation is not clear. Thus, there remains a need for the identification and characterization of additional mutants and polypeptides encoded thereby involved in these pathways of plant growth.